Lysophosphatidic acid (LPA) is a naturally-occurring phospholipid having the simplest chemical structure, and it has been known to exhibit a growth factor-like activity against the fibroblasts [Moolenaar, W. H. et al., (1992) Rev. Physiol. Biochem. Pharmacol. 119, 47–65; Moolenaar, W. H., (1995) J. Biol. Chem. 270, 12949–12952; Moolenaar, W. H. et al., (1997) Curr. Opinion Cell Biol. 9, 168–173]. The LPA is rapidly produced and released as a product of the coagulation process of blood from the activated platelets. Therefore, it is suggested that the LPA has a kind of a role in healing of a wound and regeneration. Also, there is clarified that the LPA induces rapid retraction of the axon and transient formation of spheres of cell bodies in neurocytes. These biological activities are thought to be caused by LPA receptor conjugated with G protein [Moolenaar, W. H. et al., (1997) Curr. Opinion Cell Biol. 9, 168–173], and a cDNA encoding two deduced LPA receptors has been isolated [Hecht, J. H. et al. (1996) J. Cell Biol. 135, 1071–1083; Guo, Z. et al. (1996) Proc. Natl. Acad. Sci. USA 93: 14367–14372].
In addition, the LPA possesses various biological activities such as promotion of cancer cell invasion, cell adhesion, suppression of apoptosis, and chemotaxis. Moreover, there has been reported that its amount present in sera is high in ovarian cancer and other gynecologic malignancies [Xu, Y. et al. (1998) JAMA 280, 719–723], and it is expected to also serve as a marker for early detection of cancers.
Regarding the production of the LPA, there has been known that the LPA is synthesized by generation from monoacylglycerol by monoacylglycerol kinase, generation from phosphatidic acid by phospholipase A1 (PLA1) or phospholipase A2 (PLA2), and the like. However, since the LPAs produced in response to various stimuli take place at a slightly delayed stage as compared to the generation of phosphatidic acid (PA) [Gait, F. et al. (1997) FEBS Lett. 410, 54–58], it is thought that those ascribed to the latter pathway are dominant.
However, the LPA in the biosynthesis system is immediately converted to phosphatidic acid, so that it is thought that most of the cases where the LPA acts as a bioactive substance is not made from the synthesis system but as a product of a phospholipid degradation system. Regarding the hydrolytic pathways of the LPA, there are possibly three pathways, namely pathways with LPA phospholipase A, LPA phosphatase and LPA acyltransferase [Gait, F. et al., (1997) FEBS Lett. 410, 54–58; Eberhardt, C. et al., (1997) J. Biol. Chem. 272, 20299–20305]. Since the LPA is a bioactive lipid, it is thought that the exclusion of the LPA by the above enzyme plays an extremely important role in termination of the signal.
Regarding LPA phospholipase A, one purified from rat brain has been known, which is a membrane bound enzyme of a size of 80 kDa, and hydrolyzes LPA but does not hydrolyze other lysophospholipids [Thompson, F. J. and Clark M. A., (1994) Biochem. J. 300, 457–461].
Regarding the LPA phosphatase, there has been reported the presence of ecto-(lyso)phosphatidic acid phosphatase that also hydrolyzes PA in PAM212 mouse keratinocytes [Xie, M. and Low, M. G., (1994) Archives Biochemistry Biophysics 312, 254–259]. Until recently, a membrane bound PA phosphatase which is relatively PA-specific but also exhibits a weak activity for LPA has been purified from porcine thymus [Kanoh, H. et al., (1992) J. Biol. Chen. 267, 25309–25314; Kai, M. et al., (1996) J. Biol. Chem. 271, 18931–18938]. In addition, a membrane bound PA phosphatase, which also hydrolyzes LPA, ceramide 1-phosphate and sphingosine 1-phosphate, is purified from rat liver [Waggoner, D. W. et al., (1995) J. Biol. Chem. 270, 19422–19429; Waggoner, D. W. et al., (1996) J. Biol. Chem. 271, 16506–16509]. However, there has not yet been known LPA-specific phosphatase at present.
Further, regarding the method for determination of LPA, there has been known a method comprising extracting lipid components from a sample, separating LPA from other lipid components by thin layer chromatography, and thereafter determining the resulting LPA by gas chromatography as a methyl ester of a fatty acid via transmethylation reaction [Xu, Y. et al. (1998) JAMA 280, 719–723]. However, this method requires complicated procedures, so that it has a defect that a length of time period is required for assay of a large number of samples.